New targets are needed for detecting disease through molecular imaging (Massoud, T. F. & Gambhir, S. S., Genes Dev 17, 545-80 (2003); Herschman, H. R., Science 302, 605-8 (2003); Rudin, M. & Weissleder, R., Nat Rev Drug Discov 2, 123-31 (2003); Weissleder, R. Nat Rev Cancer 2, 11-8 (2002)) and for treating disease through directed delivery in vivo (Drews, J., Science 287, 1960-4 (2000); Lindsay, M. A., Nat Rev Drug Discov 2, 831-8 (2003); Workman, P., Curr Cancer Drug Targets 1, 33-47 (2001); Anzick, S. L. & Trent, J. M. Oncology (Huntingt) 16, 7-13 (2002); Cavenee, W. K., Carcinogenesis 23, 683-6 (2002)). Genome completion identifies a target pool of 40,000 genes which may translate into a million possible protein targets (Huber, L. A., Nat Rev Mol Cell Biol 4, 74-80 (2003)). Genomic and proteomic analysis of normal and diseased tissues have yielded thousands of genes and gene products for diagnostic and tissue assignment as well as potential therapeutic targeting (Drews, J., Science 287, 1960-4 (2000); Lindsay, M. A., Nat Rev Drug Discov 2, 831-8 (2003); Workman, P., Curr Cancer Drug Targets 1, 33-47 (2001); Anzick, S. L. & Trent, J. M., Oncology (Huntingt) 16, 7-13 (2002); Huber, L. A., Nat Rev Mol Cell Biol 4, 74-80 (2003); Perou, C. M. et al., Nature 406, 747-52 (2000)). Yet the sheer number of candidates can overwhelm the required in vivo validation process, leading some to question the ultimate impact of these approaches on speeding up drug discovery (Drews, J., Science 287, 1960-4 (2000); Lindsay, M. A., Nat Rev Drug Discov 2, 831-8 (2003); Workman, P., Curr Cancer Drug Targets 1, 33-47 (2001); Huber, L. A., Nat Rev Mol Cell Biol 4, 74-80 (2003)). Reducing tissue data complexity to a manageable subset of candidates most relevant to targeting, imaging, and treating disease is clearly desired but requires new discovery and validation strategies that effectively focus the power of global identification technologies.
Selectively targeting a single organ or diseased tissue such as solid tumors in vivo remains a desirable yet elusive goal of molecular medicine that could enable more effective imaging as well as drug and gene therapies for many acquired and genetic diseases (Massoud, T. F. & Gambhir, S. S. Genes Dev 17, 545-80 (2003); Herschman, H. R., Science 302, 605-8 (2003); Weissleder, R., Nat Rev Cancer 2, 11-8 (2002); Lindsay, M. A., Nat Rev Drug Discov 2, 831-8 (2003); Huber, L. A., Nat Rev Mol Cell Biol 4, 74-80 (2003)). Most tissue- and disease-associated proteins are expressed by cells inside tissue compartments not readily accessible to intravenously injected biological agents such as antibodies. This inaccessibility hinders many site-directed therapies (Drews, J., Science 287, 1960-4 (2000); Lindsay, M. A., Nat Rev Drug Discov 2, 831-8 (2003); Workman, P., Curr Cancer Drug Targets 1, 33-47 (2001); Jain, R. K., Nat. Med. 4, 655-7 (1998); Dvorak, H. F., et al., Cancer Cells 3, 77-85 (1991)) and imaging agents (Massoud, T. F. & Gambhir, S. S., Genes Dev 17, 545-80 (2003); Herschman, H. R., Science 302, 605-8 (2003); Rudin, M. & Weissleder, R., Nat Rev Drug Discov 2, 123-31 (2003); Weissleder, R. Nat Rev Cancer 2, 11-8 (2002)). For example, multiple barriers to solid tumor delivery prevent effective immunotherapy in vivo, despite efficacy and specificity in vitro (Jain, R. K., Nat. Med. 4, 655-7 (1998); Dvorak, H. F., et al., Cancer Cells 3, 77-85 (1991); von Mehren, M., et al., Annu Rev Med 54, 343-69 (2003); Farah, R. A., et al., Crit Rev Eukaryot Gene Expr 8, 321-56 (1998); Carver, L. A. & Schnitzer, J. E., Nat Rev Cancer 3, 571-81 (2003); Schnitzer, J. E., N Engl J Med 339, 472-4 (1998)). Conversely, the universal access of chemotherapeutics dilutes efficacy to require increased dosages leading to unwanted systemic side effects. Thus, new approaches are required that cut through the cumbersome overabundance of molecular information to permit rapid discovery and validation of accessible tissue-specific targets that can direct molecular imaging and pharmacodelivery in vivo.
Vascular endothelial cells form a barrier in vivo that can greatly limit the ability of many drugs, gene vectors, and imaging agents circulating in the blood to reach their intended target cells residing within a single tissue. This restricted accessibility can prevent therapeutic efficacy in vivo and increase therapeutic side effects. Vascular targeting is a new drug and gene delivery strategy that targets the luminal endothelial cell surface and its caveolae which are directly exposed and thus inherently accessible to agents circulating in the blood (McIntosh, D. P., et al., Proc Natl Acad Sci USA 99, 1996-2001 (2002); Carver, L. A. & Schnitzer, J. E., Nat Rev Cancer 3, 571-581 (2003)). Agents such as peptides and antibodies to endothelial cell surface proteins show promise for directing tissue-specific pharmacodelivery to the vasculature in vivo (McIntosh, D. P., et al., Proc Natl Acad Sci USA 99, 1996-2001 (2002); Pasqualini, R. & Ruoslahti, E., Nature 380, 364-366 (1996); Muzykantov, V. R., et al., Immunotargeting of antioxidant enzyme to the pulmonary endothelium. Proc Natl Acad Sci USA 93, 5213-5218 (1996); Muzykantov, V. R. et al., Proc Natl Acad Sci USA 96, 2379-2384. (1999)) but greater molecular information and more candidate targets expressed in vivo are needed to understand and define the potential of vascular targeting.
The endothelium exists as an attenuated cell monolayer lining all blood vessels, and forming a physiologically vital interface between the circulating blood and the underlying cells inside the tissue. It plays a significant role controlling the passage of blood molecules and cells into the tissue and in many other normal physiological functions including vasoregulation, coagulation, and inflammation as well as tissue nutrition, growth, survival, repair and overall organ homeostasis and function (Schnitzer, J. E., Trends in Cardiovasc. Med. 3, 124-130 (1993)). Disruption of the vascular endothelium and its normal barrier function can lead rapidly to tissue edema, hypoxia, pathology, and even organ death (Fajardo, L. F., Am. J. Clin. Pathol. 92, 241-250 (1989); Jaffe, E. A., Cell biology of endothelial cells. Hum. Pathol. 18, 234-239 (1987)).
Although the microenvironment of the tissue surrounding the blood vessels appears clearly to influence greatly the phenotype of the endothelial cells (Madri, J. A. & Williams, S. K., J. Cell Biol. 97, 153-165 (1983); Goerdt, S. et al., Exp Cell Biol 57, 185-192 (1989); Gumkowski, F. et al., Blood Vessels 24, 11-23 (1987); Hagemeier, H. H., et al., Int J Cancer 38, 481-488. (1986); Aird, W. C. et al., J Cell Biol 138, 1117-1124 (1997); Janzer, R. C. & Raff, M. C. Nature 325, 253-257 (1987); Stewart, P. A. & Wiley, M. J., Develop Biol 84, 183-192 (1981)), currently there is very little molecular information about vascular endothelium as it exists natively in the tissue. This is in large part because of technical limitations in performing large-scale molecular profiling on a cell-type that comprises such a small percentage of the total cells in the tissue. Past approaches have relied primarily on genomic or antibody-based analysis of endothelial cells isolated from the tissue by enzymatic digestion to disassemble the tissue and release single cells for sorting using endothelial cell markers (Auerbach, R., et al., Microvasc Resn 29, 401-411 (1985); St Croix, B. et al., Science 289, 1197-1202 (2000); Plendl, J., et al., Anat Histol Embryol 21, 256-262 (1992)). Over the last three decades, the study of isolated and even cultured endothelial cells has yielded much functional and molecular information; however, both the significant perturbation of the tissue and the growth in culture contribute to morphologically obvious phenotypic drift that can translate rapidly into loss of native function and protein expression (Madri, J. A. & Williams, S. K., J. Cell Biol. 97, 153-165 (1983); Schnitzer, J. E. in Capillary Permeation, Cellular Transport and Reaction Kinetics. (ed. J. H. Linehan) 31-69 (Oxford Press, London; 1997). The reported ability of specific cells and select peptides displayed on bacteriophage to home to specific tissues of the body after intraveous injection also provides indirect evidence supporting the molecular heterogeneity of endothelial cell surface in different organ (Pasqualini, R. & Ruoslahti, E., Nature 380, 364-366 (1996); Plendl, J., et al., Anat Histol Embryol 21, 256-262 (1992); Rajotte, D. et al., J Clin Invest 102, 430-437 (1998)) but have not yet facilitated mapping of endothelial cell surface proteins in vivo. The degree to which endothelial cell expression is modulated within different normal and diseased tissues remains unclear.